The activity and immune dynamics of PD-1 inhibition on high-risk pulmonary ground glass opacity lesions: insights from a single-arm, phase II trial

Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) protein significantly improve survival in patients with advanced non-small-cell lung cancer (NSCLC), but its impact on early-stage ground-glass opacity (GGO) lesions remains unclear. This is a single-arm, phase II trial (NCT04026841) using Simon’s optimal two-stage design, of which 4 doses of sintilimab (200 mg per 3 weeks) were administrated in 36 enrolled multiple primary lung cancer (MPLC) patients with persistent high-risk (Lung-RADS category 4 or had progressed within 6 months) GGOs. The primary endpoint was objective response rate (ORR). T/B/NK-cell subpopulations, TCR-seq, cytokines, exosomal RNA, and multiplexed immunohistochemistry (mIHC) were monitored and compared between responders and non-responders. Finally, two intent-to-treat (ITT) lesions (pure-GGO or GGO-predominant) showed responses (ORR: 5.6%, 2/36), and no patients had progressive disease (PD). No grade 3–5 TRAEs occurred. The total response rate considering two ITT lesions and three non-intent-to-treat (NITT) lesions (pure-solid or solid-predominant) was 13.9% (5/36). The proportion of CD8+ T cells, the ratio of CD8+/CD4+, and the TCR clonality value were significantly higher in the peripheral blood of responders before treatment and decreased over time. Correspondingly, the mIHC analysis showed more CD8+ T cells infiltrated in responders. Besides, responders’ cytokine concentrations of EGF and CTLA-4 increased during treatment. The exosomal expression of fatty acid metabolism and oxidative phosphorylation gene signatures were down-regulated among responders. Collectively, PD-1 inhibitor showed certain activity on high-risk pulmonary GGO lesions without safety concerns. Such effects were associated with specific T-cell re-distribution, EGF/CTLA-4 cytokine compensation, and regulation of metabolism pathways.


Thyroid-stimulating hormone (TSH) ≤ULN;
Bilirubin ≤ULN; ALT and AST≤1.5 ULN; AKP≤2.5 ULN; Serum creatinine ≤ 1.5 ULN or creatinine clearance≥60mL/min 6) Women of childbearing age must already be using reliable contraception or have had a pregnancy test (serum or urine) with a negative result within 7 days prior to inclusion and be willing to receive appropriate method of contraception during the trial and 8 weeks after the last trial drug administration.For males, using appropriate methods of contraception or surgical sterilization are needed during the trial period and 8 weeks after the last course of medication; 7) The participants voluntarily joined the study and signed the informed consents.The participants had good compliance and cooperated with follow-up visits.

Exclusion criteria
Patients will be excluded if they meet one of below conditions: 1) Non-calcified lesions with a diameter of > 3cm were present 2) Patients with distant metastases were excluded 3) The presence of any active autoimmune disease or a history of autoimmune disease (as follows, but not limited to: autoimmune hepatitis, interstitial pneumonia, uveitis, enteritis, hepatitis, hypophysitis, vasculitis, nephritis, hyperthyroidism, reduced thyroid function; Participants who had vitiligo or had complete remission of asthma in childhood could be included without any intervention as adults; Asthma in which participants require medical intervention with bronchodilators is not included); HBsAg positive, HBV DNA≥2000 IU/ml or copy number ≥104/ml; Hepatitis C reference: HCV antibody positive;

Exit criteria
1) The participant withdraws informed consent and requires withdrawal; 2) During the enrollment period, participants were required to undergo surgical resection of target lesions; 3) Imaging examination showed the progression of the disease; 4) Those who cannot tolerate toxicity; 5) The participants lost follow-up or had positive HCG; 6) Other situations in which the investigator deems it necessary to withdraw from the study.Termination criteria 1) Finding that there is an unexpected, significant or unacceptable risk to the participant; 2) The investigational drug/trial treatment does not work, or it is pointless to continue the trial; 3) The applicant decides to terminate the study due to reasons such as significant delay in participant inclusion or frequent protocol deviations.

Primary endpoint:
Objective response rate (ORR) Secondary endpoint: The safety of sintilimab

Sample size
According to previous studies, the ORR of anti-PD-1 antibody monotherapy for non-small cell lung cancer (NSCLC) was assumed to be 20%, and the ORR of routine observation was not more than 5%.According to the optimal design principle of Simon Phase II trial, α=0.05 (bilateral), β=0.2, the sample size of phase I was calculated to be 10 cases.If no one case was effective, the trial was terminated.If 1 case or more is effective, then enter the second stage, continue to enroll 19 cases, combined with the first stage, a total of 29 cases, according to the 20% of dropping out rate, the study needs to enroll 36 cases.

Therapeutic regimen
Sintilimab of 200mg was administered intravenously per 3 weeks, and the efficacy was evaluated after the second and fourth treatment cycles.

Statistical analysis
The Student's t-test, Wilcoxon's rank-sum test, and ANOVA were applied to compare continuous variables and the χ2 test or Fisher's exact test was applied to assess the association between categorical variables.The ORR and TRAEs were expressed as frequencies and percentages.Exact tests were performed where applicable.
A two-sided p-value <0.05 was treated as significant.

Notes:
1. ICF signing should be performed prior to any protocol-specified procedures, except for tumor imaging and tumor tissue biopsy within the specified time limit prior to the first dose.
2. Previous medications include initial diagnostic therapy, including chemotherapy, radiotherapy and surgery, etc., and the time of the last anti-tumor therapy before that must be recorded.
3. Vital signs include temperature, pulse rate, respiratory rate, and blood pressure.It was performed during the screening period, before each sintilimab administration, at the end of treatment, and at the safety follow-up.Blood pressure monitoring: During each blood pressure measurement, smoking and coffee drinking should be prohibited within 30 minutes before the measurement, and at least 10 minutes of quiet rest should be taken.During the measurement, the elbow should be placed in the sitting position at the same level as the heart, and each blood pressure measurement should be taken on the same side.Blood pressure was measured by the investigator during the screening period and before each planned sintilimab infusion.During the study, blood pressure monitoring was completed by the participants themselves and recorded in the participants' diary card.Blood pressure was measured at least 3 times a week in the first 2 cycles, and if blood pressure was abnormal, it was tracked every day.If the blood pressure was normal, the blood pressure was checked twice a week after the third cycle.
4. Height measurements were performed only during the screening period.Weight measurements were required during the screening period, before each dose, at the end of treatment, and at safety follow-up visits.
5. Physical examinations were performed during the screening period, before each sintilimab administration, at the end of treatment, and at safety follow-up visits.
6. ECOG PS was performed during the screening period, before each sintilimab administration, at the end of treatment, and at the safety follow-up.
7. 12-lead ECG was performed during the screening period, within 30 minutes of completion of each sintilimab infusion, at the end of treatment, and at safety follow-up visits.
8. Blood routine includes: red blood cell (RBC), hemoglobin (HGB), hematocrit value 23.Safety follow-up will be conducted at 90±7 days after the last dose.
24. Survival follow-up: telephone visits were taken once every 8 weeks (±7 days) after the last dose.In the process of tumor occurrence and development, tumor cells have many genetic and epigenetic changes compared with normal cells, and theoretically have enough antigens to be recognized by the human immune system, and then trigger an immune response to inhibit tumor growth.In fact, tumors evade the immune system by suppressing the immune response against tumors in a variety of ways.With the deepening understanding of the body's immune system and the rapid development of biotechnology, immunomodulatory therapy has become an important means of cancer treatment, and occupies a more and more important position in the comprehensive treatment system of cancer.

Abbreviation
PD-1 (programmed cell death protein-1), a molecule originally thought to be associated with cell death.With the deepening of research, scientists found that PD-1 is not related to programmed cell apoptosis, but has the function of negative regulation of immunity.PD-1 is an inhibitory receptor mainly expressed on T cells.Under normal physiological conditions, PD-1 can inhibit the activation of T cells and the production of cytokines by binding to its two ligands (PD-L1 / PD-L2), thereby protecting the body from the attack of the autoimmune system.Tumor cells can successfully evade recognition and attack by the body's immune system through the combination of these PD-L1 molecules and PD-1 on T cells.Anti-PD-(L)1 antibody can block this 'tumor immune escape mechanism' and restore the anti-cancer activity of the patients' immune system.
Anti-PD-1 antibody has been successfully used in many types of cancer, including lung cancer, and has become a standard treatment choice for the first and second line.
Some advanced patients have achieved durable remission and long-term survival.In recent years, PD-1 inhibitor has also achieved excellent results in earlier neoadjuvant therapy, with a significantly higher response rate than advanced patients.Combined with the principle of immunotherapy, it is generally believed that PD-1 inhibitor is more effective in early tumors.Therefore, this study aimed to explore the efficacy of PD-1 inhibitor on multiple primary lung cancer in the early stage.
Clinical implications of this study: given the remarkable efficacy of immunotherapy in advanced NSCLC and the urgent need to treat unresectable nodules in patients with MPLC, it is promising to investigate the utility of PD-1 inhibitors on early-stage GGO lesions.

Primary endpoint:
Objective Response Rate (ORR)

Secondary endpoint:
The safety of sintilimab

Study design
This is a single center, single-arm, phase II study using the Simon's optimal two-stage design

Sample size
According to previous studies, the ORR of anti-PD-1 antibody monotherapy for nonsmall cell lung cancer (NSCLC) was assumed to be 20%, and the ORR of routine observation was not more than 5%.According to the optimal design principle of Simon Phase II trial, α=0.05 (bilateral), β=0.2, the sample size of phase I was calculated to be 10 cases.If no one case was effective, the trial was terminated.If 1 case or more is effective, then enter the second stage, continue to enroll 19 cases, combined with the first stage, a total of 29 cases, according to the 20% of dropping out rate, the study needs to enroll 36 cases.

Inclusion criteria
1) Above 18 years of age; 2) Two or more GGO lesions (pure GGO or GGO-predominant) that cannot be resected simultaneously; 3) There was at least one lesion with a diameter of 1-3cm pathologically confirmed as lung cancer; Serum creatinine ≤ 1.5 ULN or creatinine clearance≥60mL/min 6) Women of childbearing age must already be using reliable contraception or have had a pregnancy test (serum or urine) with a negative result within 7 days prior to inclusion and be willing to receive appropriate method of contraception during the trial and 8 weeks after the last trial drug administration.For males, using appropriate methods of contraception or surgical sterilization are needed during the trial period and 8 weeks after the last course of medication; 7) The participants voluntarily joined the study and signed the informed consents.The participants had good compliance and cooperated with follow-up visits.

Exclusion criteria
Patients will be excluded if they meet one of below conditions: 1) Non-calcified lesions with a diameter of > 3cm were present 2) Patients with distant metastases were excluded 3) The presence of any active autoimmune disease or a history of autoimmune disease (as follows, but not limited to: autoimmune hepatitis, interstitial pneumonia, uveitis, enteritis, hepatitis, hypophysitis, vasculitis, nephritis, hyperthyroidism, reduced thyroid function; participants who had vitiligo or had complete remission of asthma in childhood could be included without any intervention as adults; Asthma in which participants require medical intervention with bronchodilators is not included); IU/ml or copy number ≥104/ml; Hepatitis C reference: HCV antibody positive.

Exist criteria
1) The participant withdraws informed consent and requires withdrawal; 2) During the enrollment period, participants were required to undergo surgical resection of target lesions; 3) Imaging examination showed the progression of the disease; 4) Those who cannot tolerate toxicity; 5) The participants s lost follow-up or had positive HCG; 6) Other situations in which the investigator deems it necessary to withdraw from the study.

Termination criteria
1) Finding that there is an unexpected, significant or unacceptable risk to the participants; 2) The investigational drug/trial treatment does not work, or it is pointless to continue the trial; 3) The applicant decides to terminate the study due to reasons such as significant delay in participant inclusion or frequent protocol deviations.

Treatment regimen and dosage
200mg sintilimab, intravenous drip, 3 weeks as a treatment cycle, the efficacy was evaluated after 2 courses of treatment.The drug will be discontinued owing to the occurrence of PD, intolerable toxicity, death, withdrawal of informed consent from patients.

General guidelines for dose adjustment
✓ Reasons for dose adjustment or delay, measures taken and results should be recorded in the patient's medical record and electronic case report form (CRF); ✓ If concomitant symptoms are present at baseline, the investigator will determine whether the dose will be adjusted according to the level of change in adverse effects.
For example, if a participant has grade 1 lethargy at baseline and grade 2 lethargy during the study treatment, this means that the participant has increased his grade 1 lethargy and the dose should be adjusted according to grade 1 toxicity; ✓ If there are several adverse effects of different grades or severity at the same time, dose adjustment will be based on the highest observed grade; ✓ If dose adjustment is required only because of laboratory hematological abnormalities, the dose will be adjusted according to the hematological test values before the start of the treatment cycle; ✓ Continuation of the current dose without dose adjustment or discontinuation of treatment if the adverse effect is judged by the investigator to be unlikely to develop further serious or life-threatening events.In addition, there will be no dose adjustment or suspension of treatment for anemia (non-hemolytic), which can be relieved by blood transfusion.

Dose adjustment of sintilimab
No increase or decrease in the dose of sintilimab was allowed, only suspension or termination was allowed.
For the dose adjustment of sintilimab, refer to Table 1; for the infusion reactions related to sintilimab, refer to Table 2.
In addition, investigator-assessed PD according to RECIST v1.1 criteria (unless the participant meets the criteria for continuation of treatment after progression, the drug should be permanently discontinued).

Table 1. Provisions for dose adjustment of sintilimab
Adverse events (AEs) Severity

Grade 1
The infusion rate can be reduced by 50% or the infusion can be temporarily interrupted until the infusion reaction is relieved.✓ Radiation therapy (Note: Radiation therapy may be approved and permitted by the sponsor for symptomatic isolated lesions or to the brain as long as it is not a lung or target lesion); ✓ Live vaccines including but not limited to: measles, mumps, rubella, chickenpox, yellow fever, rabies, BCG, typhoid (oral) vaccine administered within 28 days prior to the first dose of study medication and during study participation.Allow receipt of inactivated vaccine against seasonal influenza by injection; Intranasal live attenuated influenza vaccine (e.g.FluMist®) is not allowed; ✓ Corticosteroids, which allow inhaled steroids to be used as part of regular treatment for asthma or chronic obstructive pulmonary disease (COPD).Corticosteroids used to manage potential irAEs are permitted.Physiological doses of corticosteroids are 4) Patients who have used other anti-tumor drugs in clinical study within 4 weeks; 5) Patients with severe allergic reaction to monoclonal antibody; 6) Clinical symptoms or diseases of the heart that are not well controlled, such as: a. NYHA grade 2 or above heart failure; b.Unstable angina pectoris; c.Myocardial infarction within 1 year; d.Clinically significant supraventricular or ventricular arrhythmias requiring treatment or intervention; 7) Participants with congenital or acquired immune deficiency (such as HIV infected persons), or active hepatitis (hepatitis B reference:

(
HCT), white blood cell (white blood cell), WBC), platelet (PLT), leukocyte classification (lymphocyte count (LYM), absolute neutrophil count, ANC), monocyte count (MONO), eosinophil count (EOS), basophil cell count (BASO)].Blood biochemistry includes: Liver function [serum total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate transferase (aspartate transferase, AST, γ-glutamyl transferase (γ-GT), alkaline phosphatase (ALP), albumin (ALB), total protein (TP), TP), lactic dehydrogenase (LDH)]; Renal function [blood urea nitrogen (BUN), creatinine (Cr)]; Blood electrolytes (Na, K, Cl, Mg, Ca, P); Lipase, amylase and fasting blood glucose(FBG).Routine urine tests include: PH, urinary albumin (UALB), urine protein (UPRO), urine red blood cell (URBC), urine glucose (UGLU).Screening urine routine showed negative urine protein was eligible for inclusion.Stool examination: occult blood, such as fecal occult blood + must be reexamined, reexamined fecal occult blood +, require gastrointestinal endoscopy.Routine blood tests, blood biochemistry, urine tests, and stool tests were performed during the screening period (within 7 days before the first dose of the study drug), before each dose of sintilimab, at the end of treatment, and at the safety follow-up.Inspections will be conducted at the various research centers.9. Coagulation tests include: thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time, APTT) and international normalized ratio (INR).It was performed within 7 days before the first dose of study treatment, before the administration of sintilimab b on day 1 of each cycle, at the end of treatment, and at the safety follow-up.Inspections will be conducted at the various research centers.10.Women of childbearing age will have a serum pregnancy test within 3 days before the first dose and at the end of treatment.Inspections will be conducted at the various research centers.11.Thyroid function tests include: triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (free thyroxine 4, T4), FT4) and thyroid stimulating hormone (TSH).It was performed during the screening period, each cycle 2-4, at the end of treatment, and at the safety follow-up.Inspections will be conducted at the various research centers.12.Myocardial enzyme spectrum examination: LDH, AST, Creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and ALT.The examination was performed once within 7 days before the first dose of study treatment, and thereafter only when symptoms such as precardiac pain, palpitations, and ECG abnormalities were present, and at the end of treatment.13.Pituitary adrenal axis (HPA) examination: including hypothalamic adrenocorticotropic hormone releasing hormone (CRH), pituitary adrenocorticotropic hormone (plasma ACTH) and adrenocortical hormone measurement.Adrenocortical hormone measurements included serum cortisol, urinary free cortisol (UFC), urinary 17-ketosteroid (17-KS), and urinary 17-ketosteroid (17-KGS).In eligible hospitals, monitoring is recommended during the screening period, before administration of sintilimab on day 1 of each cycle in cycles 2-4, and at the end of treatment.14.Including HBV, HCV and HIV antibody tests, HBV test requirements: During the screening period, HbsAg was tested to determine whether HBV infection was detected, and if positive, HbsAg (quantitative), HbsAb (qualitative), HbcAb (qualitative), HbeAg (qualitative), HbeAb (qualitative) and HBV-DNA (qualitative, if positive, quantitative testing must be performed).HCV testing requirements: During screening, test for HCV-Ab to determine HCV infection, and test for HCV-RNA if positive (qualitative, or quantitative).Hepatitis B carriers participating in the study should ask the researcher to arrange antiviral treatment at their discretion.15.Echocardiography: One examination should be performed within 7 days before the first dose and at the end of the study.This examination should be supplemented if clinically significant ECG abnormalities occur during the study.16.Pulmonary function test: Tidal volume (VT), respiratory rate (BE), resting ventilation per minute (MV), inspiratory volume (ERV), inspiratory volume (IC), maximal vital capacity (VCmax), forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), forced expiratory volume in the first second to vital capacity ratio (FEV1/FVC), Peak flow velocity (PEF), maximal expiratory flow at 75% vital capacity (MEF75), maximal expiratory flow at 50% vital capacity (MEF50), maximal expiratory flow at 25% vital capacity (MEF25), maximal expiratory flow at 25% vital capacity (MVV), Total lung capacity (TLC), residual to total ratio (RV/TLC), ventilatory reserve function (BR), pulmonary carbon monoxide dispersion capacity in one breath (DLCO SB), airway resistance (Raw eff) and specific airway conductance (sGaw eff) were performed during the screening period and subsequent according to investigator judgment.17.Safety assessment of AE and laboratory tests will be evaluated according to NCI CTCAE v4.03.AEs from the first dose of study drug to the end of safety follow-up and all SAEs from the signing of ICF to the end of safety follow-up should be recorded in the CRF.AE and SAE are defined, recorded, judged for relevance, judged for severity, reported time limit and treated as described in Part 9 of the protocol.AEs associated with study drugs need to be followed until remission to grade 0-1, symptom stabilization, participant withdrawal from ICF, or initiation of new antitumor therapy, whichever occurs first.18.The imaging methods used for tumor evaluation at baseline must be consistent with those used at each subsequent follow-up evaluation, and computed tomography (CT) or magnetic resonance imaging (MRI) scans are recommended.Other examinations are performed depend on the symptoms and signs of each participant.At baseline, chest, abdominal and pelvic scans (scanning from the apex of the lung to the suprapubic symphysis), brain and bone scans, and all known or suspected disease sites must be performed.Each subsequent clinical tumor imaging evaluation should include the chest, abdomen, and pelvis.Brain and/or bone scans may be performed when clinically indicated.Researchers can increase the frequency of imaging monitoring according to the actual situation.Baseline tumors information will be collected within 28 days before the first dose, and imaging data obtained before signing informed consent can be used for screening tumor assessment as long as protocol requirements are met.Evaluation was performed every two cycles until the end of the study or progressive disease (PD) was recorded on imaging.For participants with first documented response (CR or PR) and first documented imaging PD, an additional imaging assessment was required at 4 cycles (±7 days) to confirm response and PD.For discontinuation of treatment for reasons other than imaging PD, imaging evaluation may be performed according to the imaging evaluation time cut-off until any of the following events occur: initiation of new anti-tumor therapy, PD, withdrawal of the participant from ICF, and/or death.20.Participants are required to provide archived or fresh tumor tissue samples that meet the testing requirements during the screening period.In the case of obtaining the ICF of the participant, the surgical resection can be entered at the end of the treatment and the pathological response rate can be evaluated after the procedure.21.With the participant's ICF, approximately 16ml of peripheral blood can be taken during the screening period, each tumor evaluation period, and at the end of treatment for tumor markers.22.Termination of treatment refers to confirmation of disease progression or withdrawal from the study, which takes place within ±3 days of the decision to terminate treatment and/or withdraw from the study.
function of vital organs meets the following requirements: Absolute counting of neutrophil ≥1.5×109/

4)
Patients who have used other anti-tumor drugs in clinical study within 4 weeks; 5) Patients with severe allergic reaction to monoclonal antibody; 6) Clinical symptoms or diseases of the heart that are not well controlled, such as: a. NYHA grade 2 or above heart failure; b.Unstable angina pectoris; c.Myocardial infarction within 1 year; d.Clinically significant supraventricular or ventricular arrhythmias requiring treatment or intervention; 7) Participants with congenital or acquired immune deficiency (such as HIV infected persons), or active hepatitis (hepatitis B reference: HBsAg positive, HBV DNA≥2000 of drug administration after symptom improvement to grade 0-1 or baseline level.b: Hypophysis, adrenocortical insufficiency, and type I diabetes can be readministered when they are adequately controlled and require only physiological hormone replacement therapy.c: For grade 4 abnormal laboratory findings, discontinuation should be based on accompanying clinical symptoms/signs and on the investigators' judgment.

For
antihistamines at investigator discretion according to clinical practice; -Considering prophylaxis prior to administration of Grade 2 The infusion rate can be reduced by 50% or temporarily interrupted until the infusion reaction is relieved, and the infusion rate can be adjusted to 50% of the drugs during the study Participants were prohibited from receiving any of the following treatments for the duration of the study: ✓ Systemic chemotherapy and biologic therapies (including antineoplastic agents with immunoregulatory effects, including, but not limited to, interferons, interleukin-2, thymosin, immune cell therapies, etc.); ✓ Immunotherapy not specified in this protocol; ✓ Study drugs other than those specified in this study, including proprietary Chinese medicines with clear anti-tumor indications; Timeline 2019.09~2020.09Enroll patients, analyze data and record data; 2020.10~2022.04Follow-up and data collection of enrolled patients; 2022.05~2023.05Further analyses and exploration.